This grant is a response to RFA CA-15-008, question 1: For tumors that arise from a pre-malignant field, what properties of cells in this field can be used to design strategies to inhibit the development of future tumors? A key question we address is how the pre-malignant field specifically reshapes the epigenetic landscape of emerging cancers to promote metastatic behavior, which accounts for the majority of cancer-related deaths. Even prior to malignant transformation, the epigenome destabilizes and degrades lineage specific expression patterns. Since chromatin modifications respond to environmental cues, these early epigenetic shifts are likely to be critical in understanding how the tumorigenic microenvironment determines subsequent cancer cell phenotype. However, very few studies have addressed the process by which the pre-malignant environment broadly reshapes the epigenome. Enhancers are key elements that control cell-type specific gene expression patterns, and are activated in a coordinated fashion to determine cell lineage. We have identified novel enhancers of the SHC1 gene which drive expression of the lineage-specific isoform p66Shc, a protein that controls anoikis and functions as a strong metastasis suppressor. In metastatic lung cancer cells, we find that Aiolos, a chromatin regulator normally involved in lymphocyte lineage determination, silences p66Shc enhancers. Aiolos also silences putative enhancers of multiple adhesion-related genes besides SHC1, while also inducing lymphocyte homing receptors. In human tumors, high levels of Aiolos correlate with markedly worse survival rates. Further, we note aberrant expression of Aiolos by IHC in normal-appearing bronchial epithelium adjacent to Aiolos-positive lung tumors but not in epithelium remote from these tumors, suggesting an epigenetic field defect inherited by lung cancer cells. Finally, we replicate the induction of Aiolos in lung cancer cells through exposure to specific inflammatory factors know to both promote inflammation-associated tumorigenesis and drive lymphocyte differentiation. In this proposal, we hypothesize that such factors within an inflammatory pre-malignant field shift the enhancer landscape of lung epithelium and confer certain lymphocyte-like properties that promote lethal complications such as metastasis. In the first Aim we will define enhancer constituents of the gene for Aiolos responsive to such factors. In the second aim we will construct an epigenome-wide map of enhancers either activated or decommissioned in association with Aiolos induction, to identify patterns responsible for changes in epithelial cell phenotype. In the third aim we will compare these enhancer landscapes to those found in lung tumor epithelium in comparison with those in histologically normal lung epithelium from tumor-adjacent and remote tissues. In the fourth aim we will attempt to rewrite Aiolos- directed histone modifications through functional epigenomics. This project charts new territory in both the conceptual basis of tumor metastasis and in the design of novel therapeutic approaches against it.